Western blot assay

1. Cells were collected and washed with PBS, then RIPA buffer were used to get cell lysate.
2. Cell lysate supernatant was collected by centrifuge (12000rmp, 20min, 4­°c), 4X laemmli buffer (biorad, 161-0747) was added.
3. 4-15% mini-protean TGX gel (biorad, 4561086) was used for electrophoresis (Marker: thermo 26634).
4. Nitrocellular membrane (biorad, 162-0112) was used for membrane transfer (250mA, 90min)
5. Membrane blocking: 5% non-fat milk was used (RT, 30min)
6. Primary antibody (1:1000 dilution in PBST with 5%BSA) was incubated in cool room overnight.
7. Washing: PBST was used for washing (3 X 10min)
8. Secondary antibody was incubated at RT for 1 hour.
9. Pierce ECL western blot substrate was used for staining.

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